However, the potential for oral resveratrol supplements to mediate protective effects on OA have not been examined. Therefore, the aim of the present study was to investigate the potential anti-OA effects of oral resveratrol on mice fed a high-fat diet (HFD). C57BL/6J male mice were fed either a standard diet or a HFD, and a subset of the latter also received varying doses of resveratrol. Twelve weeks later, all of the animals were sacrificed and knee joints were evaluated with histological, immunohistochemical, and TUNEL analyses. Mice that received a HFD had significantly greater body weights than the control mice and also exhibited features consistent with knee OA. The mice that received a HFD in combination with low, intermediate, or high doses of resveratrol were only slightly heavier than the control mice at the end of 12 weeks. Quantitative histological assessments indicated that resveratrol treatment partly recovered joint structure in the mice that received a HFD, while high doses of resveratrol prevented the degradation of type II collagen into C-telopeptide of type II collagen (CTX-II) and retained type II collagen expression in cartilage. Furthermore, TUNEL analyses revealed a reduction in chondrocyte apoptosis in the resveratrol-treated mice compared with the HFD mice. Thus, oral resveratrol appears to exert anti-OA effects in a mouse model of HFD-induced OA, thereby highlighting the potential preventive and therapeutic value of administering resveratrol for obesity-associated OA.The effects of resveratrol on osteoarthritis (OA) pathogenesis have been demonstrated  Osteoarthritis (OA) is a chronic degenerative joint disease that is characterized by the erosion of articular cartilage and osteophyte formation. As a result, individuals with OA experience pain, joint deformation, and even disability. Currently, clinical management of OA focuses on relieving symptoms by reducing pain and inflammation and improving joint function. However, the evolving and pathophysiological processes underlying OA progression are not addressed. Furthermore, long-term use of available pharmacological agents to relieve OA symptoms is associated with substantial gastrointestinal, renal, and cardiovascular side effects ,2. ThereIn vitro studies have demonstrated the capacity for resveratrol to mediate OA-protective effects, including anti-apoptotic, anti-inflammatory, and anti-oxidant effects [In vivo evidence further suggests that resveratrol decreases chondrocyte apoptosis, decreases synovial nitric oxide content [in vitro or following intra-articular injections of resveratrol directly to the joint, while the potential for resveratrol to mediate OA-protective effects via oral supplementation remains to be investigated.Resveratrol is a natural polyphenolic compound that is found in grape skins, berries, and peanuts . In vitr effects ,5,6. In  content , and pre content . The effAnimal models of OA have been established in various laboratories to study the mechanisms of OA development ,13,14,15The aim of the present study was to investigate the effects of oral resveratrol on OA pathogenesis in C57BL/6J mice that were fed a HFD. To address this objective, we employed a mouse model of knee OA and evaluated its response to a HFD with and without resveratrol. The impact of resveratrol on the expression and degradation product of type II collagen and chondrocyte apoptosis were also examined.ad libitum and all of the animals received CMC or resveratrol every day for 12 weeks. Body weight and the amount of food intake were recorded each week. Mice were maintained under a constant temperature of 20 ± 2 °C, a relative humidity of 50% ± 10%, and a 12 h light/dark cycle. Animal experimental procedures were conducted according to an animal protocol that was approved by the Institutional Animal Care and Use Committee of China Medical University  with a ethic approval code of CMU62033009. Two mice (one in the RES-L group and one in the RES-I group) died during the experimental period.Seven-week-old male C57BL/6J mice were purchased from the Animal Center of China Medical University . The animals were randomly divided into five groups of 12 mice to establish a control (CON) group, a HFD group, a low-dose resveratrol (RES-L) group, an intermediate-dose resveratrol (RES-I) group, and a high-dose resveratrol (RES-H) group. Mice in the CON group received a standard diet with 10% of its kilocalories  derived from fat. Mice in the HFD, RES-L, RES-I, and RES-H groups received a homemade HFD containing 58% of its kcal from fat , while mg for 15 min and stored at −80 °C. Serum triglycerides and total cholesterol were determined using commercial kits . Serum levels of CTX-II were determined by ELISA . All experimental protocols were performed according to the manufacturers’ instructions.All mice were sacrificed after 12 weeks. Blood samples were collected from the abdominal aorta and were centrifuged at 1000× 2 for two months at room temperature (RT). After the joints underwent dehydration and paraffin embedding, serial sagittal sections (5 μm) were cut from the whole medial compartment of the joint. The sections were stained with hematoxylin and eosin (H & E) , Safranin O , or Safranin O/Fast Green  and were examined microscopically. All sections were observed and evaluated by two authors . Histological evaluations of cartilage were performed by calculating modified Mankin scores [Whole left knee joints were removed by dissection, were fixed in 4% paraformaldehyde for 24 h, and were decalcified in 10% EDTA-Nan scores  (the orin scores ). The grBriefly, tissue sections were incubated overnight at 4 °C with an anti-mouse type II collagen antibody  and then with an appropriate secondary antibody (Boster Biotechnology) at 37 °C. After 30 min, bound antibodies were visualized using peroxidase-conjugated avidin and diaminobenzidine according to the manufacturer’s instructions (Boster Biotechnology). Histomorphometric measurements were made using image analysis software , and positive cells were defined based on their type II collagen immunostaining. Cartilage areas were selected, and staining signals were quantified and averaged.TUNEL assays were performed according to the manufacturer’s instructions . Briefly, sections were incubated with proteinase K for 15 min at RT, then were washed with Tris Buffered Saline. The sections were subsequently incubated with a buffer containing TdT and DIG-d-UTP for 2 h at 37 °C in a humidified chamber, were blocked for 30 min at RT, were incubated with a biotin-conjugated anti-digoxin antibody (1:100) for 30 min at 37 °C in a humidified chamber, and were incubated in Strept Avidin-Biotin Complex (SABC) for 30 min at 37 °C. The staining of each section was examined by fluorescence microscopy and the number of TUNEL-positive cells in the cartilage above the tidemark was determined.p-value less than 0.05 was considered significant. Pearson linear regression was used to determine the degree of association between CTX-II levels, type II collagen expression, and Mankin scores. The linear regression coefficient, R2, is presented.Data are expressed as the mean ± standard error (SE). Statistical analysis was performed using one-way analysis of variance . A p < 0.01; p < 0.05; p > 0.05; p < 0.05; versus 3.0 ± 0.2 g/day in the HFD group versus 3.0 ± 0.3 g/day in the RES-L group versus 3.1 ± 0.2 g/day in the RES-I group versus 3.2 ± 0.7 g/day in the RES-H group, p > 0.05).Mice in the HFD group exhibited a significant increase in body weight between two weeks and 12 weeks after the start of the HFD diet compared with the CON mice (p < 0.05), while serum triglyceride concentrations decreased by 17% in the RES-L group, 25% in the RES-I group, and 15% in the RES-H group, compared with the HFD group. However, none of these reductions were statistically significant .The severity of OA in each group was further evaluated using a Mankin scoring system. The Mankin score for the HFD group was markedly higher compared with the CON group .CTX-II, a degradation product of type II collagen, may reflect the degree of articular cartilage damage that has occurred in the early stages of OA ,28. Therp < 0.05; Based on the immunohistochemical analyses that were performed, lower levels of type II collagen were detected in the HFD group compared with the CON group .To date, no measurable diagnostic markers for OA have been established. Therefore, in order to explore a potential method for assessing OA in our animal model, we analyzed the association between Mankin scores, serum CTX-II levels, and type II collagen expression by regression analysis. When Pearson correlation coefficients were calculated, there was no correlation observed between Mankin scores, CTX-II levels, and type II collagen expression  a HFD administered to C57BL/6 mice for 12 weeks increased the incidence of knee OA; (ii) oral supplementation of resveratrol at doses of 22.5 mg/kg and 45 mg/kg partially inhibited or delayed the development of OA by decreasing body weight, reducing degradation of type II collagen, and suppressing chondrocyte apoptosis.HFD-induced susceptibility to OA depends on mouse strain and gender . SeveralThe biological effects of resveratrol have been well documented in the literature. The therapeutic potential of resveratrol is reflected by its anti-oxidant, anti-apoptotic, and anti-inflammatory capacities, as well as its neuroprotective and cardioprotective roles ,6,31,32.In the present study, we administrated resveratrol to mice by oral gavage. This method is associated with minimal trauma and high compliance. Body weights in the resveratrol-treated groups were all significantly decreased, suggesting that resveratrol may exert anti-OA effects in obese mice partly by reducing biomechanical overloading and inflammatory factors.In addition to body weight, serum CTX-II levels and type II collagen expression were also assessed to determine the effect of resveratrol on OA. Serum levels of CTX-II have been shown to positively correlate with cartilage degeneration ,28, and In vitro, resveratrol has been shown to inhibit chondrocyte apoptosis induced by IL-1β-stimulated inflammation in human articular chondrocytes [p = 0.069) and the RES-H group (p = 0.062) were markedly lower compared with the HFD group. Taken together, these results indicate that resveratrol mediates at least a modest anti-apoptotic effect on obesity-related OA. To our knowledge, our findings are the first to demonstrate that oral resveratrol exerts anti-OA effects by decreasing body weight, by retaining type II collagen, and by reducing chondrocyte apoptosis in mice fed a HFD. Therefore, the potential for resveratrol to serve as a preventive or therapeutic strategy for obesity-related OA should be further investigated, particularly the administration of oral resveratrol. Regarding the latter, a HFD should be administered over a longer period of time, and appropriate dosing and administration schedule should be optimized.It is reported that an abundance of apoptotic cells can be found within the chondro-osteophyte at the periphery of OA joints, even in the early stages of disease . Moreovedrocytes ,39,40. Adrocytes . This redrocytes . Based oIn conclusion, our findings demonstrated that a HFD administered to C57BL/6 mice increased the incidence of knee OA, and oral resveratrol partially inhibited the development of OA by decreasing body weight, reducing degradation of type II collagen, and suppressing chondrocyte apoptosis. Thus, the administration of oral resveratrol appears to exert anti-OA effects in a mouse model of HFD-induced OA, thereby highlighting the potential preventive or therapeutic value of resveratrol for obesity-associated OA.


The adsorption characteristics of aqueous phosphorous are determined along with the essential kinetic parameters.On-site soils are increasingly used in the treatment and restoration of ecosystems to harmonize with the local landscape and minimize costs. Eight natural soils from diverse ecological zones in the source area of a drinking-water reservoir in central China are used as adsorbents for the uptake of phosphorus from aqueous solutions. The X-ray fluorescence (XRF) spectrometric and BET (Brunauer-Emmett-Teller) tests and the Scanning Electron Microscopy (SEM) and Fourier Transform Infrared (FTIR) spectral analyses are carried out to investigate the soils’ chemical properties and their potential changes with adsorbed phosphorous from aqueous solutions. The intra-particle diffusion, pseudo-first-order, and pseudo-second-order kinetic models describe the adsorption kinetic processes. Our results indicate that the adsorption processes of phosphorus in soils occurred in three stages and that the rate-controlling steps are not solely dependent on intra-particle diffusion. A quantitative comparison of two kinetics models based on their linear and non-linear representations, and using the chi-square ( The safety of drinking water supplies has been seriously threatened by water eutrophication. The primary cause of eutrophication is the oversupply of nutrients, which induces explosive growth of certain types of harmful algae and, consequently, disturbs aquatic ecosystems ,2,3,4,5.3 of water annually from the Danjiangkou reservoir on the Han River, which is a large tributary to the middle reaches of the Yangtze River, to Beijing and Tianjin. More and more studies have been carried out to preserve public health in the Han River basin and develop a water conservation strategy for SNWTP [The three-route  South-to-North Water Transfer Project (SNWTP) was implemented in 2002 to mitigate the increasing water resource deficits in north China ,9. The Mor SNWTP ,11,12,13or SNWTP . Algal bor SNWTP ,16. Theror SNWTP . In thesFor the purpose of optimally designing and controlling treatment processes of aqueous phosphorous, it is important to model its adsorption rate, which determines the residence time required for completion of the pertinent reactions, and to simulate the kinetic and dynamic behavior of pollutants on the adsorbents . SeveralThis work investigates the time-dependent adsorption characteristics of aqueous phosphorus in eight soil types having wide-ranging physicochemical properties and found in diverse representative ecological zones. The soils have potential use as ecological restoration sites in a source area for drinking water in central China, that is, the Danjiangkou Reservoir. Diffusion and kinetic models as well as adsorption rate constants are used to describe the adsorption characteristics of aqueous phosphorus. This work’s findings have significance for assessing the feasibility of using on-site natural soils in the ecological treatment and restoration pilot applications of phosphorous removal.Eight soil types were obtained from surface soils (0–20 cm deep) from different representative ecological zones in the Danjiangkou reservoir area in central China. Three to five replicate soil samples were retrieved randomly at each site. This region has a subtropical monsoonal climate. The average annual precipitation is approximately 900 mm and 70%–80% of the precipitation occurs between April and October, the mean annual air temperature ranges from 12 °C–16 °C. 2 gas BET (Brunauer-Emmett-Teller) analysis was made by using a Micromeritics Chemisorption ASAP 2020. The Scanning Electron Microscopy (SEM) measurement was carried out using a FEI Quanta 200 apparatus. Fourier Transform Infrared (FTIR) spectra were recorded in the 400 cm−1–4000 cm−1 range, and measured with a 4 cm−1 resolution, using a Nicolet 5700 FTIR spectrometer.Soil samples were air-dried, prescreened to remove large gravel particles and woody fragments, and crushed gently and sieved to particle size <0.15 mm prior to the adsorption experiments. The samples chemical composition analysis was carried out using an X-ray fluorescence (XRF) spectrometer Bruker AXS S4 Pioneer. A NA beaker (5 L) was prepared by mixing 40 g of dried soil adsorbents, which were oven-dried at 170 °C for 2 h before experiments to inhibit the microbial activity, and 1L liquid solution with initial phosphorus concentrations equal to 10 mg/L (0.02 mol/L KCl was added as electrolyte to each solution). The reaction occurred at a constant temperature of 25 °C and at a constant agitation speed of 170 rpm. Five milliliters of slurry was pipetted out at different times and centrifuged immediately for 1 min at 10,000 rpm at 25 °C in a HITACHI CR22G II High-speed Refrigerated Centrifuge and then filtered to completely separate the liquid and solid phases. The remaining phosphorus concentration was measured according to the ammonium molybdate spectrophotometric method with a spectrophotometer.r2).The kinetics of adsorption of phosphorus onto soil adsorbents were analyzed using the intra-particle diffusion, pseudo-first-order model and pseudo-second-order models. Statistical criteria used for estimating the goodness-of-fit of the models to the experimental data were the coefficients of determination (Pk is the inter particle diffusion rate constant (mg/(g·min1/2)), and C is the intercept (mg/g).The intra-particle diffusion model, developed to describe the diffusion mechanism and rate controlling steps that affect the adsorption process, is expressed by Equation (1) .(1)qt=kThe pseudo-first-order kinetic model, presented by Lagergren, is expressed by Equation (2) :(2)dqtdtq = 0 at t = 0 to tq = tq at time t yields:k1 is the rate constant of adsorption (g/min), eq is the amount of phosphorus adsorbed at equilibrium (mg/g), and tq is the amount of phosphorus adsorbed at time t (mg/g).Integrating Equation (2) between the limits from initial conditions Ho’s pseudo-second-order kinetic model is expressed by Equation (4) ,22:(4)dtq = 0 at t = 0 the integrated form of Equation (4) is:k2 is the rate constant of adsorption (g/(mg·min)), eq is the amount of phosphorus adsorbed at equilibrium (mg/g), and tq is the amount of phosphorus adsorbed at time t (mg/g).For initial conditions The chemical compositions of eight soils from diverse ecological zones in the Danjiangkou reservoir area were characterized using XRF spectrometric analyses. 2 are similar (0 (equilibrated pressure/saturation pressure) [0: 0.45). This kind of feature can be also found for other soils [2 are listed in Table S2. Most pores in the studied soil samples were sized at about 4.90–10.13 nm in diameter. The BET surface areas varied from 37.88 m2/g–1.93 m2/g, with decreasing values in the order OS > CCS > NGS > FCS > ACS > RS > BS > FS. The total pore volumes of the eight soils ranged from 0.0509 cm3/g–0.0070 cm3/g in increasing order for the eight soils in the sequence CCS > FCS > NGS > OS > ACS > RS > BS > FS. Soil OS had the largest BET surface area and soil CCS had the largest total pore volume, while soil FS had the smallest BET surface area and total pore volume.To gain further insights into the porous nature of the studied soil adsorbents, BET measurements were performed to examine their surface textural properties. Although all eight soils were derived from diverse ecological zones, their adsorption-desorption isotherms for N similar . Generalressure) . Moreoveer soils . The porThe surface morphology and chemical make-up features of soil samples and their potential changes with the adsorption of phosphorus were also investigated. Before and after the adsorbent phosphorus was extracted from aqueous solution, soil CCS  was selected as an example of the soil samples and examined by SEM and FTIR analyses. It was found that the original soil sample had a rough surface with irregularly distributed gaps a,b. The −1 accompanied by OH stretching vibrations at 3425–3621 cm−1. A weak band near 1432 cm−1 is characteristic of calcium carbonate. The broad band around 1031 cm−1 can be assigned to Si-O stretching vibrations. The double-peak bands near 779 cm−1 are assigned to the symmetric stretching vibration of O-Si-O in soil. The Si-O-Moct  bending vibrations bands appear within the 400–600 cm−1 region [−1 indicate that those microstructures may be involved in the extracting of phosphorus during the adsorption process.The FTIR spectrum of the CCS soil sample  shows ob1 region . The phoThe effect of contact time on the amount of phosphorus adsorbed was investigated in a batch reactor with rapid stirring. The extent of removal of phosphorus by all eight soil adsorbents was found to increase and then gradually approach a more or less constant value with the increase in agitation time. Similarly, a rapidly increasing adsorption in the first 10 min for each soil adsorbent was observed, and the uptake of phosphorus after 20–40 min approached a constant asymptotic value, denoting attainment of equilibrium. However, the initial adsorption rates of eight soil adsorbents varied. Therefore, the intra-particle diffusion, pseudo-first-order, and pseudo-second-order models were introduced in this study to investigate their adsorption kinetics and test the adsorption mechanism subsequently.The adsorption process for porous solid adsorbents can be described by four steps: (1) transport in the bulk of the solution; (2) external binding or boundary layer diffusion; (3) intra-particle diffusion; and (4) adsorption and desorption within the particle and on the external surface ,28,29,30tq) onto different soil adsorbents against the square root of the time of uptake (t1/2) was investigated. If the intra-particle diffusion was involved in the adsorption process, the plot of t vs. tq1/2 would yield a straight line, and, if the rate-limiting step was only intra-particle diffusion, this line would pass through the origin [t vs. tq1/2 for eight soil adsorbents are shown in The diffusion mechanism and rate-controlling steps that affected the adsorption process were explored with the intra-particle diffusion model . The amoe origin ,32. The idk, are obtained from the slopes of the lines in stage 2 (idk is minimum for soil NGS (0.0058 mg/g·min1/2) and maximum for soil CCS (0.0146 mg/g·min1/2) with the experimental adsorption setup. As shown in C, which are associated positively with the boundary layer effect, are also calculated from the second, linear portions of the plots of t vs. tq1/2 [t vs. tq1/2 by the eight soil adsorbents near the origin are obvious. It may be concluded that the adsorption mechanism of phosphorus on soils from diverse ecological zones in Danjiangkou reservoir area is complex and that the adsorption process is not solely rate-controlled by the intra-particle diffusion step.The values of the rate constant for intra-particle diffusion,  stage 2 . The valvs. t1/2 ,35. GeneThe pseudo-first-order and pseudo-second-order models are commonly compared and applied in their linearized forms, and the least squares method is always used for determining the best-fitting models and obtaining the kinetic parameters. . The pseeq − tq) versus t for the adsorption of phosphorus onto eight soils were tested. Linear fitness examination of the pseudo-first-order rate plots and the calculated r2 equal to 0.8050, 0.8760, 0.6445, 0.5970, 0.6852, 0.5978, 0.8187, or 0.7658 confirmed that the adsorption kinetics did not fit well with this model for soils OS, FS, NGS, ACS, FCS, CCS, RS, or BS, respectively. The pseudo-second-order model (Equations (4) and (5)) can be also linearized as four different types, and the constants of the model are calculated experimentally by plotting t/t versus tq, 1/t versusq 1/t, t versus qtq/t, and tq/tt versus q for linear equation type 1 (Equation (7)), type 2 (Equation (8)), type 3 (Equation (9)), and type 4 (Equation (10)), respectively [The plots of log(ectively .(7)tqt=qe, k2, and the r2 for four expression types were significantly different. Linear fitness examination of the pseudo-second-order rate plots revealed that the type 1 linear equation yielded the best results with high r2 values (0.9951–0.9999) (χ2) test was used as the statistical criteria to quantitatively compare the applicability of each kinetic equation, and as the r2 for estimating the goodness-of-fit of the models to the experimental data [–0.9999) a. The catal data ,38. The r2 and low χ2 values, for all eight soil adsorbents obtained using the non-linear method, indicate that the pseudo-second order kinetic model gives an agreement between the experimental and calculated adsorption capacity values that is better than that obtained with the linear model. The comparison results between the pseudo-first-order model and the pseudo-second order model obtained with linear and non-linear methods is consistent: the pseudo-second order model is more suitable for the description of the kinetic nature of phosphorus adsorption on each soil adsorbent. The experimental data of soil CCS was fitted best with the pseudo-second order model, with highest r2 and lowest χ2 of 0.9948 and 0.0008 by the non-linear method, and 0.9999 and 0.0011 by the linear method.r2 and the χ2 values obtained with the linear method were larger than those obtained with the non-linear method (r2 and χ2 being simultaneously larger with the linear method than with nonlinear method) might be caused by the transformation of the non-linear pseudo-second-order kinetic model to its linear form. This kind of transformation might implicitly alter the model’s error structure and violate the error variance and normality assumptions of the standard least squares method [r2 was significantly influenced, but the χ2 test avoided this error [χ2 values, even though the values of phosphorus adsorbed at equilibrium  calculated with the linear method were closer to the experimental  values  values .k2 and qe, are plotted against the main surface textural characteristics of the soil adsorbents in versus k2 , BET surface area versus qe , and total pore volume versus k2 , total pore volume versus qe , indicate that the adsorption rates and capacities of phosphorus are directly proportional to the porosity of the studied soil adsorbents. Therefore, high adsorption rates and capacities could be obtained on those fine-textured soils offering active adsorption sites in the Danjiangkou reservoir area.The adsorption rate and capacity may be affected by the physical and chemical properties of the adsorbent, such as the porosity . The non-linear pseudo-second-order kinetic constants, The time-dependent adsorption behavior of phosphorus for eight soils, collected from diverse representative ecological zones in the source area of drinking water of a reservoir in central China, was investigated in this paper. A similar elemental composition and surface textural nature was found for all eight soils. No significant change in the surface morphology and chemical make-up features of the studied soil with the addition of phosphorus was observed.2r and χ2) was caused by the transformation of the non-linear kinetic model to its linear form. This anomaly could be avoided if the chi-square (χ2) test were used as the sole statistical criterion for comparing different linear and non-linear kinetic equations.The adsorption processes of phosphorus for all eight soils exhibited three stages, instead of a rate-controlled process by intra-particle diffusion. A contradiction between diagnostic criteria  with decreasing order RS < FS < BS < ACS < FCS < OS < CCS < NGS, and the simulated adsorption capacities ranged from 0.060–0.195 mg/g with decreasing order FS < BS < RS < OS < FCS < ACS < CCS < NGS. Moreover, it was found that the adsorption rate and capacity of phosphorus were directly related to the porosity of the soil adsorbents. This result suggests that fine-textured soils have definite potential as substrate materials used in the ecological treatment and restoration systems for the removal and retention of phosphorus.


Several 3D multi-segment foot models (MFMs) have been introduced for the in vivo analysis of dynamic foot kinematics. However, reproducibility of a model should be checked and ascertained before clinical utilization of a MFM. The purpose of this study was to determine the reliability of recently introduced MFM with a 15-marker set by assessing the participant’s stride-to-stride (intra-session) and visit-to-revisit (inter-session) repeatability.Twenty healthy adults with a mean age of 28.9 years  were tested. Three representative strides from five separate trials were used for analysis from each session. Kinematic data of foot segmental motion was collected and tracked using the Foot3D Multi-Segment Software . A retest was performed in the same manner at an interval of 4 weeks. Coefficients of multiple correlation (CMC) and intra-class correlation coefficient (ICC) were calculated in order to assess the intra-session and inter-session repeatability.Inter-segment foot angles from healthy adults from a MFM with 15-marker set showed a narrow range of variability during the gait cycle. The mean intra-session ICC was 0.981 (±0.010), which was interpreted as excellent. The mean intra-session CMC was 0.948 (±0.027), which was interpreted as very good repeatability. The mean inter-session ICC was 0.886 (±0.047) and the mean inter-session CMC was 0.801 (±0.077), which were interpreted as excellent and good repeatability, respectively.We demonstrated a MFM with a 15-marker set had high intra-session and inter-session repeatability, especially in sagittal plane motion. We thought this MFM would be applicable to evaluation of the segmental foot motion during gait. The characterization of foot mechanics during the gait cycle in healthy and diseased humans has been a challenge. In trials using three-dimensional 3D) analysis of opto-reflective markers, a ‘gold standard’ method to represent the actual motion of the tarsal bones might be the use of intra-cortical bone markers ,2, althoD analysiThese models differ in the number of foot segments analyzed, the position of markers within each segment, and the mathematical interpretation of segmental motion, leading to different segmental motion patterns during gait cycle . For exaRecently, Henley et al. proposed a 3D MFM of a 15-marker set with the goal of improving clinician’s ability to accurately implement the model in a clinical setting . This moThe purpose of this study was to determine the reliability of a 3D MFM with a 15-marker set by assessing the participant’s stride-to-stride (intra-session) and visit-to-revisit (inter-session) repeatability.2 (range 16.8-32.2). The mean spine-malleolar distance (SMD) was 86.0 cm (range 76.7-96.0). The mean foot length was 24.5 cm (range 21.5-26.9) and the mean foot width was 9.7 cm (range 8.6-11.0). Demographic data are presented in Table This study was approved by the institutional review board of Seoul National University Hospital and all participants gave informed consents prior to participation. Twenty healthy adults aged 20–35 years were tested at the Laboratory of Human Motion Analysis in Seoul National University Hospital. Volunteers were recruited from the local area with equal numbers of males and females. Inclusion criteria were 1) no history of fracture or surgery on the lower extremities; 2) no subjective symptom during gait; 3) no abnormal findings on foot radiograph; 4) no history of cardiac or respiratory disease or uncorrected visual impairment; and 5) in normal function of the foot and ankle (AOFAS ankle-hindfoot score of 100). The alignment and range of motion of the lower extremity joints  were clinically evaluated by authors  to exclude abnormal condition of the lower extremities. The mean age was 28.9 years (range 20-35) and the mean weight was 66.5 kg (range 44.9-105.5). The mean height was 168.5 cm (range 154.3-181.5) and the mean BMI was 23.3 kg/mWhen evaluating segmental foot motion, we used 15 opto-reflective markers on the anatomical landmarks of each knee, tibial shank, ankle and foot. This system consisted of 6 additional markers per foot in addition to the conventional Cleveland clinic lower extremity marker set . PlacemeFive markers were placed around the knee and tibial shank for calculation of the shank coordinate system. Four markers were placed on the ankle and hindfoot , 2 markers on the midfoot (navicular and cuboid) and 4 on the forefoot  Figure  8]. Mor. Mor8]. After explaining the procedures and obtaining written consent, we measured each participant’s demographic data including height, body weight, leg length, foot length and width. The participants performed a 5-minute warm-up protocol walking comfortably. After warming up, the reflective markers were attached to foot and lower extremities of each participant. Participants were asked to walk at a comfortable speed along an 8 m walkway. We captured a static standing trial with the individual in anatomic position. After static calibration, kinematic gait data were collected using 12 cameras with a 3D optical motion capture system  at a sample rate of 120 Hz. Eva Real-Time software  was used for real-time motion capture and for post-processing and tracking of marker data. Three representative strides from five separate trials were used for analysis from each session. A Retest was performed with the same protocol at an interval of 4-weeks to evaluate the inter-session repeatability.Kinematic data of foot segmental motion was collected and tracked using the Foot3D Multi-Segment Software . The definition of the coordinate systems based on these markers and the method used to calculate the joint rotation and arch parameters had been described previously .We divided the gait cycle into 100 time points  for data analysis. Seventeen parameters were tested for repeatability. Hallux , hindfoot , forefoot , medial forefoot , lateral forefoot  and arch parameters  were evaluated.Coefficients of multiple correlation (CMC) and intra-class correlation coefficient (ICC) were analyzed in order to assess the intra-session repeatability. Intra-session CMC and ICC were only analyzed for the first sessions. Intra-session CMC was evaluated from the first 2 strides in the selected 3 strides of the first session. Intra-session ICC evaluations were based on the selected 3 strides.For inter-session repeatability, CMC and ICC were analyzed. Data from the three trials for each visit were averaged, and then the inter-session CMC and ICC were evaluated.The difference between the two sessions was assessed for each time points of the gait cycle. After that, the average, standard error, and range of the inter-session difference were calculated.The range of motion (ROM) in each segmental foot motion was calculated in each participant. For the assessment of intra-session and inter-session repeatability of the ROM measurements, intra-session and inter-session ICC was analyzed. The intra-session ICC was evaluated based on the selected 3 strides of the first session and the inter-session ICC based on the average of each session.We interpreted that 0.65 ≤ CMC (R) < 0.75 suggests moderate repeatability, 0.75 ≤ CMC (R) < 0.85 suggests good repeatability, 0.85 ≤ CMC (R) < 0.95 suggests very good repeatability, and CMC (R) ≥ 0.95 suggests excellent repeatability . We inteThe mean cadence was 113.5 (steps/min) and the mean speed was 124.1 (cm/sec). The mean stride length was 131.1 cm and the mean step width was 10.9 cm. Generally, the patterns of segmental movement seemed to be consistent. Inter-segment foot angles from healthy adults of a MFM with 15-marker set showed a narrow range of variability during the gait cycle  is presented in Table The range of motion in hallux flexion/extension and hindfoot flexion/extension were largest in all segmental motions, which were 39.7° and 22.2°, respectively. The mean intra-session ICC of ROM measurements in 17 segmental motion parameters was 0.992 (±0.004). Intra-session ICC values of ROM in all parameters were interpreted as excellent. The mean inter-session ICC was 0.940 (±0.028). Inter-session ICC values of ROM in all parameters were interpreted to have excellent repeatability.We demonstrated that a MFM with a 15-marker set in absence of medial and lateral calcaneal markers had high intra-session and inter-session repeatability, which we believe can be applied to evaluation of inter-segmental foot motion during gait in healthy participants.The positions and motions of the foot and ankle during gait are complex in the process of supporting the body, transferring forces from the ground and adapting to uneven surface . A standIt is obvious that increasing number of markers with accurate placement will enable more precise analysis of actual segmental foot motion. However, placing multiple markers in a small area of interest might cause significant variability in marker placement. For example, in OFM, mean inter-session difference of hallux motion was highest  among all parameters . In MiFMThe model that was used in this study was a MFM adding 6 markers placed on the foot in addition to Cleveland Clinic lower extremity marker set. This model involves ten markers placed on prominent anatomical points around the foot and ankle with notable absence of medial and lateral calcaneal markers. The prominent difference of this model from other previous foot models is that this model has no medial and lateral calcaneal markers and only one hallux marker, which is placed on the the middle of the hallux nail bed. In this model, the calcaneus’ medial-lateral axis is established by the cross product of the vector from the distal marker to the ankle joint and the vector from the distal marker to the heel marker. A single vector from the 1st metatarsal head to the dorsal hallux marker is analyzed for hallux motion. We used this model based on the fact that marker placement on the medial and lateral calcaneus can be problematic  althoughWhen assessing the quantitative kinematic data of joint motion, measurement errors and variability are thought to come from three primary sources: (1) participant, (2) measurement system, and (3) examiner . NaturalIn this study, we tried to evaluate intra-session  repeatability, which is considered to reflect variability from gait variation within a participant and from the system measurement accuracy, and inter-session (between-day) repeatability, which can be considered to reflect the summation of all possible sources of error. We did not measure the between-examiner repeatability in this study. The between-examiner repeatability is considered to be mainly related with errors of marker placement. In several articles designed to evaluate between-examiner repeatability ,16, all Considering between-participant variability, in most clinical laboratories, patient gait data is compared to the average response of “normal” control participants . The conFor the statistical analysis of repeatability, the CMC, which is the positive root of the adjusted coefficient of multiple determinations (CMD) has been used extensively to evaluate similarities between kinematic waveforms ,28. The Some researchers, however, recommended including measures of absolute measurement error (e.g. SD and standard error of the mean), in order to fully appreciate reliability data ,14,16. WDespite promising results for further utilization of a present MFM, limitations of this study may be addressed as follows. First, we are still concerned that defining the hindfoot anatomical coordinate system using just two posterior calcaneal markers and the ankle joint center in this system can be problematic. The single marker placement on the hallux also may be insufficient to represent three dimensional motion of the hallux. Whether this system can be applied to patients with foot deformity or disease should be clarified further. Secondarily, we did not assess between-examiner repeatability in this study. We agree that an experienced technician is a prerequisite to increase inter-session repeatability. However, because marker placement was performed by a single technician without previous experience of MFM, we could not assess inter-rater repeatability. Finally, we were not able to compare our results directly with the previously reported studies, because there are no previous reports on the reliability of the present model.In this study, we used a MFM with 15-marker set in order to evaluate segmental foot motion in healthy adults. We demonstrated that this model had high intra-session and inter-session repeatability in the assessment of foot motion. We believe that this 3D MFM can be applicable in clinical settings, which require further elucidation.The authors declare that they have no competing interests.SGS, DYL and IHC conceived of the study; participated in design of the study; performed the data analysis including statistical analysis; and participated in drafting the manuscript. HJM, JHK, KML and CYC participated in its design and coordination and helped to draft the manuscript. SJK helped to perform statistical analysis. All authors read and approved the final manuscript.


In particular, clusters containing little information were detected with reliability. This strategy, named “cluster-aided MCR-ALS,” will facilitate the attainment of more reliable results in the metabolomics datasets.There is an increasing need to use multivariate statistical methods for understanding biological functions, identifying the mechanisms of diseases, and exploring biomarkers. In addition to classical analyses such as hierarchical cluster analysis, principal component analysis, and partial least squares discriminant analysis, various multivariate strategies, including independent component analysis, non-negative matrix factorization, and multivariate curve resolution, have recently been proposed. However, determining the number of components is problematic. Despite the proposal of several different methods, no satisfactory approach has yet been reported. To resolve this problem, we implemented a new idea: classifying a component as “reliable” or “unreliable” based on the reproducibility of its appearance, regardless of the number of components in the calculation. Using the clustering method for classification, we applied this idea to multivariate curve resolution-alternating least squares (MCR-ALS). Comparisons between conventional and modified methods applied to proton nuclear magnetic resonance ( An incorrect choice can lead to loss of information (underestimation) or the inclusion of noise components (overestimation). Many methods have been proposed for determining the number of components, including the Kaiser criterion27291H-NMR spectra data using multivariate curve resolution-alternating least squares (MCR-ALS), we were faced with this problem. A wide range of different “optimal” numbers of components had been estimated by eight different methods  was calculated (C × St). Because a cluster consists of a set of matrices (C × St), it is considered to be three-dimensional data  and the corresponding spectral profile  based on a bootstrap strategy to estimate sampling error. In this study, clusters were selected by the pvclust function with an approximately unbiased (AU) P-value of >0.95, therefore the uncertainty of the result was less than 5%. In some cases, clusters contained undesired elements that showed little similarity to other elements within the cluster. To resolve this problem, a correlation coefficient matrix was calculated. If the minimum value was under 0.6, the cluster was rejected. Pvclust was re-executed against the rejected cluster to remove undesired elements, and the correlation coefficient was confirmed again. This process was repeated until the minimum correlation coefficient was greater than 0.6. To determine the minimum cluster size, a dataset, randomly shuffled to destroy biological information, was used. We repeated the pvclust calculation with the shuffled dataset five times. We chose the maximum cluster size for each dataset and set the threshold for the cluster size as >5. As a result, there were 15 selected clusters. Clusters and their elements (components) are listed in Using the same dataset, cluster-aided MCR-ALS was performed. The MCR-ALS calculation was repeated, changing the number of components from one to 20. The total number of resulting components was 210. All concentration profiles were collected into one dataset on which cluster analysis was performed. To select the clusters with objectivity, we used “pvclust” developed by Suzuki A comparison of the results of conventional and cluster-aided MCR-ALS is summarized in Two clusters were assigned to trehalose . Aged groups were also added. In total, 18 groups of NMR spectra were analyzed, concurrently, for each urine and feces sample (see Methods section).To apply the cluster-aided MCR-ALS method to biological samples, we collected Unlike the practice in general atherosclerosis studies, the HFD was fed to mice for only 4 days, given that metabolomic changes occur quickly in response to food changes. It was unnecessary to induce atherosclerosis in the mice, because our purpose was only to evaluate this new method. Additionally, feeding HFD for only a short period conserved both time and funds in this experiment.At the outset of sample collection, 90 mice (3 strains × 2 sexes × 3 conditions × 5 mice) were available. However, one mouse, belonging to the DBA/2J female aged group, died of unknown causes. All other mice were healthy during the experimental period. Sample collection and NMR analysis were performed successfully. The final number of samples was 89.1H-NMR spectroscopy studies performed in mice have been reported. Our urinary spectra data showed a pattern similar to that of previous reports, which include signals from acetate, citrate, creatine, creatinine, hippurate, lactate, taurine, trimethylamine (TMA), trimethylamine N-oxide (TMAO), and other metabolites. . We identified 29 metabolites in fecal extracts, including short-chain fatty acids  and branched-chain amino acids  and 2,550 (feces) after components with all elements zero were removed. All concentration profiles were combined into one dataset on which cluster analysis was performed. Cluster selection was performed in the same way as standard mixture analysis except that analysis of shuffled data was repeated 10 times for determining the minimum cluster size. Finally, we identified 21 (urine) and 35 (feces) reliable clusters. Clusters and their elements (components) are listed in Concentration profiles for both methods of urinary analysis are shown in 1H-NMR chemical shifts of taurine were 3.26 and 3.43 ppm. The signals were confirmed by 1H-13C-heteronuclear single quantum coherence (HSQC)-NMR. From the result of cluster-aided MCR-ALS, both taurine signals were observed in cluster 144_5 . The TMAO signal was accordingly assigned as either 3.26 or 3.30 ppm because of variation in experimental conditions such as sample temperature, pH, and ionic strength. The TMAO signal was observed in cluster 92_1_2 (3.30 ppm) and cluster 117_4  in male HFD-fed mice . In convThe color-coded bar graph in In PCA, the importance of a principal component is assessed by its eigenvalue or contribution. These parameters are based on the amount of information in each component; in this strategy, components with large amounts of information are preferentially chosen over components with less information. Many methods for determining numbers of components are also based on the amount of information. However, although there is no evidence to suggest that all biologically informative components contain large amounts of information; biologically informative components with small amounts of information are not detected by conventional methods. In biological systems, large changes may be triggered by small changes534In the urinary data analysis, clusters 67 and 92_1_2 showed a high sum of squared deviations. These patterns were observed in both cluster-aided MCR-ALS and the conventional method  in the HFD-fed group in both conventional and cluster-aided MCR-ALS. TMA is produced from choline by gut microbiota and converted to TMAO by flavin-containing monooxygenase form 3 (FMO3) in liver43P-value of >0.95, whereas the final number of retained clusters was 21. In a similar manner, 231 clusters were identified and 35 clusters were retained in the fecal data analysis. In both datasets, approximately 85% of clusters were considered unreliable. We cannot yet explain why unreliable components comprise a large fraction of total clusters. Unreliable clusters may reflect a loss of precision caused by an excess of variables. In theory, the number of samples is larger than the number of variables. However, in many cases, this is not practical in the analysis of omics datasets. Another factor causing this is the threshold values for selecting reliable clusters. Two threshold values are important; one is the AU P-value, estimated by the R package pvclust. We used 0.95 as a threshold value. Higher values, such as 0.97, 0.98, or 0.99 should be tested to optimize analytical conditions. The other threshold value is the size of the cluster, which reflects the reproducibility of the component. A large size means a highly reproducible component. To determine the threshold size, pvclust was performed with a randomly shuffled dataset. A reliable cluster size should be larger than the threshold size. In principle, this process should be repeated many  times. However, we repeated the analysis only 10 times  because of limited access to a high-performance computing environment. It is also necessary to optimize the clustering algorithm, which may be Euclidean, maximum, or Manhattan for the distance matrix, or methods such as Wards, single, complete, and median for cluster forming. Because this calculation must be repeated many times, high-performance computer resources such as a supercomputer should be used.In the urinary analysis, pvclust initially identified 151 clusters with an AU 1H-NMR datasets as a test case. Concentration and spectral profiles of identified reliable clusters showed more plausible patterns than the results of conventional MCR-ALS. Cluster-aided MCR-ALS avoids the need to determine the number of components prior to the analysis, a requirement of the conventional method. This report has shown that cluster-aided MCR-ALS is a feasible method for analysis of 1H-NMR datasets. Cluster-aided MCR-ALS will also be applicable to other omics data. The algorithm can also be applied to other methods such as ICA/NMF. Optimization of the conditions and speed of the calculation will be necessary for further development of the cluster-aided MCR-ALS strategy.In summary, we introduced the idea of “reliable” and “unreliable” components based on the reproducibility of components in repeated MCR-ALS calculations with the number of components changed for each calculation. We evaluated this strategy, named “cluster-aided MCR-ALS,” using urinary and fecal 1H-NMR spectroscopy, 3-(trimethylsilyl)-1-propanesulfonate sodium salt (DSS), was purchased from Sigma-Aldrich Japan. Stable isotopically labeled substrate, 13C6-D-Glucose, was obtained from Cambridge Isotope Laboratories, Inc., USA.The internal standard for ad libitum access to water and conventional chow diet, CA-1 .All animal experiments were approved by the Animal Research Committee of the RIKEN BioResource Center and were performed in accordance with RIKEN guidelines for animal experiments. Three laboratory mouse strains, C57BL/6J, C3H/HeJ, and DBA/2J, were purchased from a commercial breeder  and maintained in our facility . All mice were maintained at constant room temperature (23 °C ± 2 °C) and humidity (55 ± 10%) under a light/dark cycle of 12/12 h in a specific pathogen-free (SPF) environment. The pathogens were specified in categories A and B of the International Council for Laboratory Animal Science. Mice were weaned between the ages of 4 and 5 weeks. Mice had 13C6-D-glucose water, ad libitum. After habituation for 4 days (from the evening of day 1 to day 4), urine and feces were collected within a time span of 18 h from the evening of day 4 to the morning of day 5.Laboratory mice were divided into three experimental groups, “control,” “HFD feeding,” and “aged,” from which all NMR samples were collected. The control group consisted of 30 14-week-old mice, both females and males, of three strains (five mice from each sex of each strain). On day 1, the mice were placed into the metabolic cage unit  in the evening. Mice had access to ND, which is a conventional chow diet (CA-1) and 2% For the HFD-feeding group, 16-week-old mice were used. Except for the use of a HFD, the number of mice and sampling procedures were the same as the control group. The period of HFD feeding was approximately 4 days (evening of day 1 to morning of day 5).Sampling was performed in the same way for the aged group as for the control group. However, for the aged group, 60-week-old mice were used for urine and feces collection.The HFD used in this study was custom-designed based on CA-1 feed , and named HFD-RIKEN. It was composed of 70% CA-1, 18.55% granulated sugar, 10% cocoa butter, 1.25% cholesterol, and 0.2% cholate.2O), and 1 mM sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used as an internal standard. After centrifugation, the extracted supernatant was transferred into a 5-mm Φ NMR tube. All one-dimensional (1D) Watergate spectra were acquired at 298 K on a DRX-500 spectrometer , operating at 500.13 MHz and equipped with a 1H inverse triple-resonance probe with triple-axis gradients (Bruker Biospin), as previously described1H-13C-HSQC) spectra were recorded on a Bruker DRU-700 NMR spectrometer equipped with a 1H inverse cryogenically cooled probe with a z-axis gradient as previously described47484952Urine and feces extract samples were suspended in 10% (v/v) deuterium oxide (D1H-NMR spectra data (0–10 ppm) obtained was binned with 0.04-ppm (urine) and 0.02-ppm (feces) intervals to result in datasets of 250 (urine) and 500 (feces) variables. The region of the peak of DSS  was eliminated. For urine spectra, the region from 4.5 ppm to 6.25 ppm was excluded to eliminate the signals from both water and urea. Each spectrum was normalized to a total intensity.The series of The MCR-ALS method was used to resolve multiple component responses in unknown mixtures54ALS was installed. Before MCR-ALS was performed, PCA was performed for estimation of the initial concentration matrix. PCA was performed using the prcomp R function. The PCA score  was used for the initial concentration matrix. For the initial spectral matrix, all values were set to 1.where E is a residual. Matrices C and S are estimated by an optimization algorithm based on a bilinear model with constraints of non-negativity. For analysis of MCR-ALS, the R package P-values for the uncertainty of each cluster using bootstrap resampling. Pvclust was applied using the average method and a correlation-based distance matrix. The bootstrap sample number was set to 1,000. The package “snow” was used for parallel computing for high speed performance of the pvclust function. The clusters that had high AU P-values (>0.95) were picked using the “pvpick” function. If some of the clusters had inclusion relationships, only the largest cluster was selected (max.only = TRUE) to avoid overlap.For cluster-aided MCR-ALS, the calculation for MCR-ALS was repeated, changing the number of components from one, sequentially, to the maximum number of components. Because the PCA score was used for the initial matrix, the maximum number of components was the same as the number of samples. Estimated concentration profiles were combined into one dataset  and applied to the clustering process. The R package pvclust was used for clusteringTo determine the threshold size of the cluster, a randomly shuffled dataset, randomized-CList-data, was constructed. Because randomized-CList-data lacks biological information, clusters are formed by chance. The randomly shuffled dataset was constructed by the “sample” function of R, and pvclust and pvpick (AU >0.95) were performed. The randomized data clustering was performed 10 times for each urinary and fecal dataset. The maximum size of the cluster was used as the threshold cluster size.Sometimes a cluster contains unrelated elements that show little similarity with other elements. To confirm the similarity among elements in the cluster, a correlation coefficient matrix was estimated. If the minimum value of the correlation coefficient was less than 0.6, this cluster was clustered again to divide it into smaller clusters, and then the correlation coefficient matrix was estimated again. This process was repeated until the minimum correlation coefficient was greater than 0.6 or the size of the cluster was below the threshold value determined by randomized data analysis. Selected clusters were considered “reliable clusters.”t was calculated for each component  TheHow to cite this article: Motegi, H. et al. Identification of Reliable Components in Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS): a Data-Driven Approach across Metabolic Processes. Sci. Rep.5, 15710; doi: 10.1038/srep15710 (2015).


C. jejuni with ganglioside-mimicking lipooligosaccharides (LOS), is an immune-mediated disorder in the peripheral nervous system (PNS) accompanied by the disruption of the blood-nerve barrier (BNB) and the blood-cerebrospinal fluid barrier (B-CSF-B). Biomarkers of GBS have been extensively explored and some of them are proved to assist in the clinical diagnosis and in monitoring disease progression as well as in assessing the efficacy of immunotherapy. Herein, we systemically review the literature on biomarkers of GBS, including infection-/immune-/BNB, B-CSF-B, and PNS damage-associated biomarkers, aiming at providing an overview of GBS biomarkers and guiding further investigations. Furthermore, we point out further directions for studies on GBS biomarkers.Guillain-Barré syndrome (GBS), the axonal subtype of which is mainly triggered by  Guillain-Barré Syndrome. Guillain-Barré syndrome (GBS) is an immune-mediated disorder in peripheral nervous system (PNS) and experimental autoimmune neuritis (EAN) serves as a main animal model of GBS. GBS is typically triggered by antecedent infections and C. jejuni is blamed for at least one-third of these infections. Nevertheless, only one in 1,000–5,000 patients with Campylobacter enteritis will develop GBS [elop GBS  and GBS  C. jejuni strains contains lipooligosaccharides (LOS), a kind of carbohydrate structure located on the outer membrane, which mimic the gangliosides in human. Autoantibodies that cross-react with gangliosides are provoked by antecedent infections and attack the PNS by activating complements [The cardinal step in the development of GBS is exerted by the immune response. A subset ofplements . Furtherplements . Cytokinplements . Nerve bplements .Damage to the PNS and the barriers, including the blood-nerve barrier (BNB) and the blood-cerebrospinal fluid barrier (B-CSF-B), is the pathological feature of GBS. BNB and B-CSF-B are barriers between blood and nerve/CSF that maintain a relatively stable environment to nerve/CSF. Distinct types of peripheral nerves damage address GBS as a highly diverse spectrum of clinical manifestations. A rapidly progressive, symmetrical weakness of the limbs in combination with hyporeflexia of areflexia is the clinical character of GBS . Some ofOverview of Biomarkers for GBS. A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of a physiological as well as a pathological process or pharmacological response to a therapeutic intervention. The diagnosis of the GBS is still challenging due to the lack of a single specific diagnostic test, which in some cases leads to a delay in the correct diagnosis and hence in the initiation of immunomodulatory treatment against GBS. The diagnosis of GBS is rather based on a combination of clinical features, NCS and analysis of the CSF at present [ present . However present . Lack ofSerum, CSF, and peripheral nerve tissue are the main sources of biomarkers for GBS. It is noteworthy that the CSF is the most important source of biomarkers. Proximal nerve roots located in the subarachnoid region are floating freely in CSF and are in close contact with CSF. Therefore, the altered protein content of CSF could mirror the damage within the tissue of the nervous system . MoreoveAt present, a growing number of studies focus on biomarkers in GBS. Although Johannes Brettschneider et al. first systemically reviewed the studies of GBS biomarkers published until October 2007, they only took CSF biomarkers into consideration. A panoramic review of biomarkers in GBS is still lacking. Herein, we summarize the studies of infection-, immune-, and BNB, B-CSF-B, and PNS damage-associated biomarkers in GBS to provide an outlook of GBS biomarkers. C. jejuni strains containing ganglioside-mimicking LOS could trigger GBS and the synthesis of LOS is controlled by a set of polymorphic genes and enzymes that vary greatly between different C. jejuni strains [Only a subset of strains . C. jejuni [ C. jejuni cst-II gene that determined the structure of LOS was associated with the occurrence of GBS while the Asn51 variant was associated with MFS [ Campylobacter strains with Penner heat-stable (HS) serotypes, including HS:1, HS:2, HS:4, HS:19, HS:23, and HS:41, were overrepresented among the strains isolated from GBS patients [The gene contents of LOS loci are divided into eight classes (classes A to H). The expression of classes A, B, C, E, F, and H loci was found in GBS-associated. jejuni , 10. Thewith MFS . Moreovepatients , 13. Furpatients . C. jejuni-related GBS patients but not in healthy controls (HCs) or in patients with OIND. The frequency of production of anti-C-Dps IgG in C. jejuni-related GBS patients was significantly higher than that in C. jejuni enteritis patients without GBS (62.5% versus 9%). C-Dps was also found in serum of some C. jejuni-related GBS patients (14.8%) [A high level of C-Dps is produced to protect bacterial DNA from damage under the condition of oxidative or nutritional stress via specifically binding to the sulfatide that is important for the maintenance of the ion channels on myelinated axons and for paranodal junction formation. Recently, C-Dps was elucidated as a potential contributor to the peripheral nerve insult in GBS. After C-Dps was injected into the rat sciatic nerves, it densely binds to the myelin sheath and the nodes of Ranvier. And NCS disclosed a compound muscle action potential amplitude reduction . Anti-C- (14.8%) . C. jejuni activates the innate immune response via interacting with immunoglobulin-like receptor LMIR5, TLR 4, and sialic acid-specific receptors which are involved in the DC-mediated Th cell differentiation and B cell proliferation [LOS offeration –19. FurtγR is a family of cell-surface molecules linking humoral and cell-mediated responses. It is expressed on almost all immune cells and plays a key role in defending against pathogens. It showed that the leukocyte degranulation and phagocytosis in GBS could be induced/blocked by anti-GM1 IgG/IVIg via FcγR that are localized on Schwann cells (SCs) and perineurial cells [γR/FcRL gene polymorphism and GBS. Among others, human leukocyte FcγR genes are divided into three classes: FcγRI, FcγRII, and FcγRIII. FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb were found to be associated with the severity of GBS [Fcal cells , 21. Sevy of GBS , 23. They of GBS .∗060x, DRB∗03:01, DRB∗07:01, DRB∗01:01, DRB1∗14/DQB1∗05, and DRB1∗13/DQB1∗03 HLA genotypes were increased in GBS patients while the frequency of DR6 HLA genotype was elevated in control group [∗01, HLA-DQA1∗0301, and HLA-DQA1∗0302/HLA-DQB1∗03 were found to be related to mechanical ventilation, anti-GM1 IgG levels, and recent C. jejuni infection, respectively [55–57/ED70-71 epitopes, DRβE9V11H13 epitopes, and HLA-DRB1∗1301 allele were found to be associated with the development of AIDP while the DQβRPD55–57 epitopes were found to be associated with the protection from AIDP [∗1301-03 and HLA-DRB1∗1312, taken collectively, were increased [Studies on the relationship between the gene polymorphism of human leukocyte antigen (HLA) complex and GBS have elucidated that the frequencies of DQbeta 1ol group –27. Furtectively –30. DQBRrom AIDP , 32. In ∗01/02 and CD1E∗01/02 genotypes, A(-670)G single nucleotide polymorphism in the promoter region of CD95, TNF-α308 G/A and 857 C/T polymorphisms, mannose-binding lectin H allele/HY promoter haplotype/HYA haplotype, macrophage mediators SNPs, TCR Vβ and Vδ genes, CD95 A(-670)G SNP, TLR 4 Asp299Gly polymorphism, killer-immunoglobulin-like receptor genotype, and glucocorticoid receptor genotype were related to GBS [Gene polymorphisms of other important molecules in GBS were studied as well. It has been documented that CD1Ad to GBS , 33–39.Cytokines are polypeptides that play a fundamental role in initiating, propagating, and regulating tissue-specific autoimmune injury . They acγ has a dual role in GBS. On the one hand, to date, most studies have addressed IFN-γ with a proinflammatory role in EAN and GBS. Clinical disability of GBS patients was positively correlated with the elevated serum level of IFN-γ and was improved after treatment with intravenous immunoglobulin (IVIg) [γ could convert peripheral CD4(+)CD25(−) T cells to CD4(+)CD25(+) regulatory T cells in GBS [γ deficiency exacerbated EAN via upregulating Th17 cells despite a mitigated systemic Th1 immune response [γ.IFN-n (IVIg) . On the s in GBS  and IFN-response  defined α plays a dual role in GBS as well. The proinflammatory function of TNF-α was identified by the association between the increased serum TNF-α levels and the severity of GBS [α and TNFR1 while enhancing the levels of sTNFR1—an antagonist of TNF-α [α secretion was associated with the altered balance of different subtypes of macrophages in EAN [It seems that TNF-y of GBS . In addiof TNF-α , 43. Howof TNF-α . It was s in EAN .IL-17 and IL-22 secreted by Th17 cells play a critical role in inflammatory disease and mucosal host defense. In GBS, plasma IL-17A and IL-22 levels were markedly elevated during the acute phase and the IL-17A concentration was reduced after IVIg therapy . Both plγ, TNF-α, and IL-10 but not the clinical severity. It indicated that IL-18 may act as a coinducer of Th1 and Th2 cytokines in EAN [IL-18 was overexpressed in the nerve roots of EAN animals . EAN mics in EAN . Prelimis in EAN .β was immunolocalized on the membranes of SCs in sural nerves [β was detected in the CSF of GBS patients. For anti-inflammatory cytokine IL-4, its upregulation in the recovery phase defined it with a role in terminating EAN and GBS [β1 levels in plasma were decreased at the onset of GBS [β secreting cells was elevated in all phases of GBS [For other cytokines, IL-1l nerves  and IL-1 and GBS , 54. Sim and GBS –57. IL-6 and GBS , 59. Int and GBS . IL-12 w and GBS , 61. IL- and GBS . IL-23 m and GBS . Interest of GBS  while tht of GBS . Howevers of GBS .Additionally, some cytokines such as IL-21, IL-27, and IL-35 that play an important role in other autoimmune diseases may be the biomarkers for GBS. Further studies are needed to explore their possible roles in GBS.Complements are another group of candidates for GBS biomarkers . In the The presence of C3 in PNS was observed in both GBS and EAN. A proteomic study addressed an enhanced C3 level in CSF . ComplemChemokines are low-molecular-weight (8–14 kDa) cytokines that are involved in the directed migrations (chemotaxis) across concentration gradients and the activation of immune cells. Chemokines are classified into 4 subfamilies based on the organization of two conserved cysteine residues: CC, CXC, CX3C, and C subfamilies. Multiple lines of evidence point out that the chemokines are involved in the immune response of GBS patients.α, was postulated to facilitate the trafficking of autoreactive leucocytes across the BNB in GBS [CCL2 was expressed on SCs, infiltrating cells and blood vessels with its receptor CCR2 expressed on macrophages and lymphocytes. CCL2, the secretion of which was stimulated by TNF-B in GBS . High exB in GBS . In GBS,B in GBS .The peak number of CCL3 positive cells was seen in the sciatic nerves of EAN 14 days after onset and was correlated with the severe clinical presentations. Anti-CCL3 antibody was demonstrated to attenuate the severity of EAN and inhibit the inflammation and demyelination in sciatic nerves. CCR1 and CCR5 expressed by endoneurial macrophages with CCL5 colocalizing to axons were increased on sciatic nerves of EAN and GBS , 80. Thehttp://dx.doi.org/10.1155/2015/564098). Additionally, other immune-related biomarkers are listed in Supplementary Table 1  Brain-Derived Proteins. A combination of elevated total protein (TP) level and normal cell counts in the CSF may be regarded as the first CSF biomarker for GBS. The elevation was directly associated with the time point of performing lumbar puncture [puncture .Prealbumin is synthesized predominantly by parenchymal cells of the liver and then secreted into the plasma. However, the prealbumin in CSF originates mainly (90%) from the choroid plexus in the ventricles. The levels of the prealbumin in both plasma and CSF were elevated in patients with GBS; nevertheless, the CSF index of prealbumin was decreased . In contS100B is a calcium-binding protein originating from glial cells. It was reported to be upregulated in the CSF of GBS patients and was correlated with the GBS disability scale scores in AIDP as well as with months to recovery , 90. Cys(2) Blood-Derived Proteins. Haptoglobin is a plasma protein with haemoglobin-binding capacity and is a positive acute-phase protein that functions as an inhibitor of prostaglandin synthesis and angiogenesis. Proteomic study revealed that the expression of haptoglobin was enhanced in CSF of GBS patients [α-1-antitrypsin levels in CSF of GBS patients [patients . This repatients . Fibrinopatients , whereaspatients . Additiopatients , 91, 95.(1) Myelin Sheath-Associated Markers. Gangliosides are a group of glycosphingolipids characterized by the presence of one or more sialic acid residues in the oligosaccharide chain. Anti-ganglioside antibodies are often closely associated with clinical phenotypes and specific clinical signs of GBS. This association is likely to depend upon the diverse distribution of ganglioside antigens in the PNS. The antigens targeted are located at or near the nodes of Ranvier in AMAN and on myelin sheath in AIDP. The antibodies can activate complements that provoke the formation of C5b-9. Interestingly, excepting antibodies against single gangliosides, patients can also have antibodies against the combination of epitopes from ganglioside complexes (GSCs). Such complexes are located in specialized microdomains or “lipid rafts” in the cell membranes [embranes . In totaembranes . Seroposembranes , 110. IgNeurofascin and gliomedin are neuronal cell adhesion molecules that play a central role in the formation of nodes of Ranvier and are considered as novel target antigens in GBS. Investigations reported the detectable autoantibodies to neurofascin and gliomedin in both GBS and EAN , 112. In14–25, and connexin 32 were detected in GBS patients [α6β4 is a laminin receptor that mediates the recognition and attachment to extracellular matrix proteins in SCs and myelin. α6β4 immunoreactivity was detected in 66% of GBS patients [ Mycoplasma pneumonia infections [P2, P0, PMP22, and connexin 32 are peripheral myelin proteins, and both P2 and P0 are used to induce EAN. Antibodies to P0, PMP22, P2patients –116. α6βpatients . Anti-phpatients . Patientfections .(2) Neuron-Component-Associated Biomarkers. Neurofilaments are cytoskeletal proteins that are particularly abundant in large myelinated axons. Their release into the CSF addressed them as a promising biomarker for neurodegeneration. The CSF neurofilaments levels were increased in GBS patients, positively correlated with the GBS disability scale scores in AMAN, and predicted the clinical/electrophysiological outcomes of GBS [s of GBS , 120. Mos of GBS .Tau proteins modulate assembly and stability in axonal damage marker. Tau concentrations in CSF from GBS patients were enhanced. The elevated tau levels were correlated with the GBS disability scale scores in AMAN and predicted poor clinical outcomes . 14-3-3 Additionally, other biomarkers that have not been included above but are related to GBS are presented in Supplementary Table 2.highCD138lowCXCR4low immature plasma cells was also observed in GBS patients. Further studies are warranted to explore more therapeutic biomarkers in GBS [Specific biomarkers are still lacking to help make exact diagnosis and predict the outcomes of GBS. A growing number of studies focus on the biomarkers of GBS and address infection-, immune-, and BNB, B-CSF-B. and PNS damage-associated molecules as potential biomarkers for GBS. Serum, CSF, and peripheral nerves are the main sources of biomarkers. Many of these biomarkers are proved to be associated with the pathogenesis, development, and recovery of GBS. IVIg treatment, inhibiting the Fc-mediated activation of immune cells as well as the binding of autoimmune antibodies to their targets, is one of the first-line immunotherapies of GBS. IVIg downregulated Th17, Th22, IL-17, and IL-22 in GBS patients and mediated expansion of regulatory T cells , 122. Cls in GBS . HoweverSupplementary Table 1: An intricate network of immune system plays an important role in the pathogenesis and the development of GBS. Except the classic immune factors including cytokines, complements and chemokines, many other molecules are found involving in the pathogenesis of GBS due to their immune effect, including erythropoietin, heat shock protein, apolipoprotein E, C-reactive protein, neopterin, matrix metalloproteinases, reactive oxygen species, cell adhesion molecules, microRNA-155 and osteopontin. They are described in detail in the table.Supplementary Table 2: Some of the biomarkers are beyond the classification set in the review. Additional information of them are provided in the table.


Synthetic L-ascorbic acid (vitamin C) is widely used as a preservative and nutrient in food and pharmaceutical industries. In the current production method, D-glucose is converted to L-ascorbic acid via several biochemical and chemical steps. The main source of L-ascorbic acid in human nutrition is plants. Several alternative metabolic pathways for L-ascorbic acid biosynthesis are known in plants. In one of them, D-galacturonic acid is the precursor. D-Galacturonic acid is also the main monomer in pectin, a plant cell wall polysaccharide. Pectin is abundant in biomass and is readily available from several waste streams from fruit and sugar processing industries.Aspergillus niger for the conversion of D-galacturonic acid to L-ascorbic acid. In the generated pathway, the native D-galacturonate reductase activity was utilized while the gene coding for the second enzyme in the fungal D-galacturonic acid pathway, an L-galactonate consuming dehydratase, was deleted. Two heterologous genes coding for enzymes from the plant L-ascorbic acid pathway – L-galactono-1,4-lactone lactonase from Euglena gracilis (EgALase) and L-galactono-1,4-lactone dehydrogenase from Malpighia glabra (MgGALDH) – were introduced into the A. niger strain. Alternatively, an unspecific L-gulono-1,4-lactone lactonase (smp30) from the animal L-ascorbic acid pathway was introduced in the fungal strain instead of the plant L-galactono-1,4-lactone lactonase. In addition, a strain with the production pathway inducible with D-galacturonic acid was generated by using a bidirectional and D-galacturonic acid inducible promoter from the fungus. Even though, the lactonase enzyme activity was not observed in the resulting strains, they were capable of producing L-ascorbic acid from pure D-galacturonic acid or pectin-rich biomass in a consolidated bioprocess. Product titers up to 170 mg/l were achieved.In the present work, we engineered the filamentous fungus In the current study, an L-ascorbic acid pathway using D-galacturonic acid as a precursor was introduced to a microorganism for the first time. This is also the first report on an engineered filamentous fungus for L-ascorbic acid production and a proof-of-concept of consolidated bioprocess for the production. L-Ascorbic acid (L-AA), also known as vitamin C, is a six-carbon organic compound with reducing agent properties. It occurs naturally in many animal and plant cells having biological functions, such as being an antioxidant and enzyme cofactor ,2. SynthGluconobacter oxydans. The second step is a mixed fermentation with Ketogulonicigenium vulgare and Bacillus megaterium, converting L-sorbose to 2-keto-L-gulonic acid which is then chemically converted to L-AA.Industrial L-AA production has been traditionally based on the Reichstein process which is an efficient multi-step and mostly chemical manufacturing method converting D-glucose to L-AA . In the K. vulgare, the bacterium that is used in the two-step fermentation process, is capable of converting D-sorbitol, L-sorbose, L-sorbosone and L-gulose directly to L-AA . Pr. PrAsper to L-AA ,20. In tA. niger strain ΔgaaB with the disrupted L-galA dehydratase gene, which is known to accumulate L-galA, was used as a platform strain. In addition, two heterologous genes from the plant or animal L-AA pathway, an ALase and a GALDH, were introduced to the strain. Several different A. niger ΔgaaB based strains with an ALase (EgALase or smp30) and a GALDH (MgGALDH) or only the GALDH were generated and tested  and a D-galUA inducible (PgaaA/C) promoter were compared.The fungal catabolic D-galUA pathway and the proposed D-galUA originating biosynthetic L-AA pathway in plants share the same reduction step yielding L-galA in the first reaction Figure . Thus, ted Table . The MgGgaaB-Eg-Mg (PgaaA/C) with D-galUA inducible EgALase and MgGALDH expression was generated using the bidirectional gaaA/C promoter. Since there are no reports on the use of gaaA/C promoter in heterologous expression, the functionality of the D-galUA inducible transcription of EgALase and MgGALDH from the gaaA/C promoter was tested using RT-qPCR  which had a higher transcription when compared to gaaA growing in D-galUA medium [EgALase had the same orientation at gaaA/C promoter as gaaC gene in the wild type strain.To avoid unnecessary and energy wasting expression of the L-AA pathway genes in non-producing conditions, the strain ΔR Figure . The traA medium . EgALasegaaB and from the further engineered strains that were cultured 20 h in the medium supplemented with 20 g l−1 D-galUA and 5 g l−1 D-xylose  strain, the GALDH activity was lower; however, it was still significantly higher if compared to the values from ΔgaaB .Enzyme activities of the introduced L-AA pathway genes were measured from crude extracts. The GALDH activity assay was based on the measurement of reduced cytochrome C as a result from oxidation of L-galL. The activity was tested from the parental strain Δe Figure . All the405 = 0.003 per 10 μM of protons which should provide high enough sensitivity for the ALase assay. We tested all the engineered strains for ALase activity in the presence of L-galA; however, we could not detect this activity.In the lactonization of L-galA to L-galL, which is catalysed by an ALase, protons are absorbed in an equimolar manner increasing the pH in the reaction. The relation between absorption at 405 nm and proton concentration was quantified being ∆AgaaB and the different engineered strains were tested for L-AA production even though the lactonase activities were not detectable. We used flask cultures on defined medium supplemented with D-galUA and D-xylose and with the initial pH adjusted to 3.0. With the strain ΔgaaB only L-AA concentrations of below 3.5 mg l−1 were observed in the cultures  with the introduced EgALase gene. In fact, the L-AA production corresponds to the GALDH activity in the strains having MgGALDH under PgpdA  had the highest titer at 96 h. The decreasing L-AA concentrations during the time course may be due to the instability of the product causing by the exposure to air. In addition, the production turned out to be pH dependent since L-AA production was not observed when the initial pH was 5.0 (data not shown).The parental strain Δs Figure . All thegaaA in the first reaction in the pathway. In addition, energy and carbon sources are needed for the expression of heterologous L-AA pathway genes. To overcome these possible limitations and to test L-AA production from a pectin-rich residue, we used CPW as substrate containing a complex mix of different sugars and sugar acids in the form of polysaccharides. We decided to use ΔgaaB-Eg-Mg (PgaaA/C) strain in order to avoid unnecessary and energy wasting expression of the heterologous genes at the beginning of fermentation when free D-galUA is not yet available. In addition, we used higher fungal biomass in the inoculation in order to ensure CPW hydrolysis and speed up the process. L-AA production from CPW turned out to be more efficient than in defined media conditions with respect to the final concentrations  strain the highest titer (around 170 mg l−1) was achieved at 96 h as was the case of the cultures in the defined conditions. As in the case of defined conditions, a decrease in L-AA concentration was observed after 96 h probably due to the instability of the product. The CPW used as a substrate did not contain detectable amounts of L-AA. In the control fermentations with the parental strain ΔgaaB, the observed L-AA concentrations were below 15 mg l−1.Even though, D-xylose was used as a co-substrate, one of the reasons for limited L-AA production may be the lack of NADPH for the D-galUA reductase s Figure . A lag p−1 that was achieved in this work is the highest among engineered fungal strains. In addition, the pathway for L-AA biosynthesis originating from D-galUA was for the first time introduced to a heterologous host. In the previous literature, L-AA production was reported to be achievable from D-glucuronic acid and D-galUA using wild type strains of A. niger [Synthetic L-AA is a widely used compound in several industries. Currently it is produced in a multistep process including chemical and biochemical steps. This study is the first report on an engineered filamentous fungus for direct L-AA production. To the best of our knowledge, the L-AA titer of 170 mg lA. niger  or yeastA. niger . HoweverA. niger .M. glabra (acerola), which is known for its vitamin C rich fruits, including the native N-terminal mitochondrial signal peptide. The kinetic parameters of MgGALDH are not known but the expression level of MgGALDH is known to be higher in M. glabra tissue when compared to the corresponding GALDH gene (AtGALDH) expression in Arabidopsis thaliana [AtGALDH gene codes for a protein having 75.25% homology with MgGALDH and kinetic parameters of Km 0.17 mM and kcat 134 s−1 for L-galL [AtGALDH gene in the A. niger ΔgaaB strain, however, L-AA was not observed in the resulting strains (data not shown). In plants, the GALDH locates on the inner membrane of mitochondria binding non-covalently FAD as a cofactor [E. coli as a cytoplasmic protein [A. niger. Mitochondrial inner membrane proteins are most often targeted to their location by an N-terminal cleavable peptide [AtGALDH and MgGALDH, the estimated signal peptides differ being 102 and 84 residues long, respectively [MgGALDH may be more suitable for mitochondrial targeting in A. niger, however, the localization of MgGALDH in the engineered A. niger strains was not investigated in this study.We decided to use the GALDH from thaliana . AtGALDHr L-galL . We havecofactor . During  protein . The res peptide . The tar peptide . The cle peptide . In the ectively . The shoEgALase or smp30 expressing strains. In addition to A. niger strains, we have also tried to express both of the ALases in yeast or E. coli strains, however, without detectable activity. The lactonase assay that was used in this work is based on a pH change and a pH indicator in the reaction mixture. The assay should provide high enough sensitivity for the activity and it has been routinely and successfully used in many studies. Consequently, the most likely explanations for the non-detectable ALase activity are inactivity of the protein in heterologous hosts or inactivation during cell disruption. In the literature, EgALase was produced at low temperature (15°C) in an E. coli strain co-expressing the Trigger Factor chaperone protein [E. coli co-expressing two chaperones GroEL and GroES [E. coli without chaperones led up to the insoluble and inactive proteins and formation of inclusion bodies [Despite several attempts, the ALase activity for L-galL could not be detected from any of  protein . In the nd GroES . In addin bodies ,30. ThusA. niger strains with a functional GALDH were capable of L-AA production. The lactonization reaction might be spontaneous and may take place extracellularly. In the lactonization reaction, protons are assimilated, which mean that a low pH shifts the equilibrium towards the lactone form. This would explain the fact that only low pH enabled L-AA production. Thus it is possible that L-galA is first secreted out from the cells and spontaneously formed L-galL is then transported back to the cells and oxidized to L-AA. On the other hand, the parental strain ∆gaaB is known for its higher capacity to produce L-galA at low pH [Regardless of the non-detectable ALase activity, several t low pH ,20 whichA. niger strains to redirect the D-galUA pathway to L-AA synthesis. The native catabolic D-galUA pathway was disrupted and the biosynthetic L-AA pathway from plants originating from D-galUA was introduced. In addition, we built a strain having the L-AA pathway under D-galUA inducible expression. The resulting strains were capable of L-AA production from pure D-galUA but also from CPW that is a pectin-rich biomass residue. Final L-AA titers up to around 170 mg l−1 were achieved with the engineered strains.We have engineered Aspergillus niger strain ATCC 1015 (CBS 113.46) was used as a wild type. The engineered strain of ATCC 1015 with the deleted L-galA dehydratase gaaB [E. coli TOP10 cells and the homologous recombination for the plasmid construction was carried out in the S. cerevisiae strain ATCC 90845.The ase gaaB ,20 was uA. niger, the heterologous genes coding for a GALDH or an ALase, listed in Table gpdA promoter. Alternatively, the GALDH from Malpighia glabra (MgGALDH) and the ALase from Euglena gracilis (EgALase) were both expressed under the native bidirectional promoter (657 bp) of the A. niger D-galUA reductase (gaaA) and the 2-keto-3-deoxy-L-galactonate aldolase (gaaC) genes that are clustered in the genome. In the expression cassette, MgGALDH was orientated as gaaA and EgALase as gaaC in A. niger genome. Terminators that were used in the bidirectional cassette for MgGALDH and EgALase were the native gaaA and gaaC terminators, respectively, both 505 bp downstream from the stop codon. All of the expression cassettes contained hygromycin B phosphotransferase (hph) gene under the gpdA promoter for the selection of transformants.For introducing the biosynthetic L-AA pathway in SacI and XmaI (both NEB) and ligated to the JKp1 plasmid. The resulting plasmids JKp1-EgALase/Smp30/MgGALDH were linearized with SpeI (NEB). For constructing the cassette with D-galUA inducible expression, the bidirectional gaaA/C promoter and terminators of gaaA and gaaC genes were amplified from A. niger ATCC1015 genomic DNA with the primers P1-P6 as described in Table EgALase, MgGALDH and the hph gene (from JKp1) were amplified with the primers P7-P12 (Table EcoRI and BamHI digested platform plasmid pRS426 were transformed to yeast using the Gietz method [EcoRI and BamHI (both NEB) before A. niger was transformed.The codon optimized ORFs were released with z method  and the A. niger ΔgaaB strain by protoplast transformation method [−1 hygromycin B . Strains having both genes (smp30 or EgALase + MgGALDH) expressed in JKp1 under gpdA promoter were generated by co-transformation. Integration of the transformed cassettes into the genome was confirmed with colony PCR using Phire direct PCR kit (Thermo Scientific) and the primers P26 and P27  was used and after transformation SCD-URA plates (uracil deficient synthetic complete media supplemented with 20 g D-glucose l−1) were used for uracil auxotrophy selection. Yeast cultures were carried out at 30°C and all the liquid cultures at 250 rpm. For the A. niger submerged fermentations, spores were generated on potato-dextrose plates and 0.9*108 spores were inoculated into 50 ml of YP medium  containing 30 g gelatin l−1. Mycelia were pre-grown in 250-ml Erlenmeyer flasks by incubating overnight at 28°C, 200 rpm and harvested by vacuum filtration, rinsed with sterile water and weighted. In the A. niger transformations and fermentations, A. nidulans defined minimal medium [−1) 6 NaNO3, 0.52 KCl, 0.52 MgCl2 and 1.52 KH2PO4 was used. In the transformations the minimal medium was supplemented with 1.2 M D-sorbitol, 10 g l−1 of D-glucose and 20 g l−1 of agar and the pH was adjusted to 6.5. The minimal medium used in the fermentations for L-AA production, enzymatic assays and transcriptional analysis was supplemented with 20 g l−1 of D-galUA and 5 g l−1 of D-xylose or with 40 g l−1 of dried citrus processing waste (CPW). The pH was adjusted to 3. The CPW contained peel and pulp from the processed oranges and was received from the Federal University of Paraná in Curitiba, Brazil. The submerged cultures supplemented with pure D-galUA and D-xylose were inoculated with 1 g l−1 (cdw) and the submerged cultures supplemented with CPW were inoculated with 3 g l−1 (cdw) of pre-grown mycelia.Luria Broth (LB) culture medium supplemented with 100 μg mll medium , containA. niger mycelia were harvested by vacuum filtration. Crude extracts were prepared in 100 mM sodium phosphate buffer pH 7 with protease inhibitor . The cells were disrupted with 0.4 mm diameter glass beads using a bead beater  and solid residues were removed by centrifugation. The GALDH assay was based on the cytochrome c reduction at 550 nm as described by Leferink et al. [For enzymatic assays, k et al. . Crude ek et al. . The pH gaaA, EgALase, MgGALDH and actin (ATCC 1015 200483-mRNA) were quantified using the primers P28-P35 (Table Samples of 2 ml were collected from the cultures and the mycelium was harvested by vacuum filtration. The filtered mycelium was frozen with liquid nitrogen and stored at −80°C. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) and ~1 μg of total RNA was used for cDNA synthesis with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche), following the manufacturer’s instructions. cDNA samples were diluted with RNase free water (Roche) and were used for RT-qPCR using a LightCycler II with the LightCycler SYBR green I Master mix (both Roche). The expression of 2SO4 as eluent and a flow rate of 0.5 ml min−1. The column was maintained at 55°C. Peaks were detected using a Waters 410 differential refractometer and a Waters 2487 dual wavelength UV (210 nm) detector. The L-AA content was measured using the commercial Ascorbic Acid Assay Kit II (Sigma-Aldrich) following the manufacturer’s instructions.Samples of 2 ml were removed at intervals and mycelium was separated from the supernatant by centrifugation. The concentration of D-galUA and L-galA was determined by HPLC using a Fast Acid Analysis Column  linked to an Aminex HPX-87H organic acid analysis column  with 5.0 mM H